Construction of Modified pTRG Vector as pTRGQQ used in Bacterial Two Hybrid Assay for Protein-Protein Interaction Studies of Dengue Virus Type-2 Protease with Peptide Libraries

In molecular biology for cloning experiments, plasmid, a circular molecule is used as vector (carrier) to insert a desired gene or DNA fragment by ligating into it after digestion of both with the compatible restriction enzymes. Then by transforming with a host (generally E. coli) strain of desired traits the screening for appropriate clone is done. A plasmid is having selectable marker in the form of antibiotic resistant gene which helps a molecular biologist for the proper selection of desired clone while screening is done. Apart from having drug (antibiotic) resistance gene a plasmid also have an origin of replication and restriction sites where a gene or DNA can be inserted without interfering with plasmid replication or expression of the drug-resistance gene. For proteinprotein interaction studies, yeast two-hybrid screening is traditionally used. It is however a tedious procedure, limited by the basic biology of the yeast. Yeast grows slowly, is difficult to transform efficiently and requires unique reagents and techniques. The bacterial two-hybrid system moves two-hybrid studies from yeast into E. coli – a much more convenient host with a fast growth rate, high transformation efficiency and easy manipulations. To carryout bacterial two hybrid assay, two plasmids are required, one which carries a gene of interest bait (pBT), and another having a target gene (pTRG). Library which is a collection of multiple DNA fragments is screened for desired clones based on blue and white color appearance on X-gal indicator plate. In this article we report construction of a new modified plasmid vector pTRGQQ derived from pTRG vector which is highly compatible for blunt end cloning at SnaB1 restriction site of target gene into it to carry out bacterial two hybrid assay.